Method for inducing bone growth

ABSTRACT

Activin is administered systemically and locally to induce the growth of mature bone. Activin enhances the level of bone formation and the quality of the bone formed when administered locally with BMP or bone marrow. Administration of activin by subcutaneous route promotes systemic increase in the bone mass.

FIELD OF THE INVENTION

The invention relates to polypeptide factors and their use in bonegrowth and maturation. Specifically, the invention relates to theisolation and purification of activin from bone and its use in bonegrowth and maturation. The invention also relates to methods of (1)locally inducing mature bone growth and maturation by administering anosteogenically effective amount of activin in combination with bonemorphogenic proteins (BMPs) and/or bone marrow; and (2) systemicallyinducing bone growth and maturation by administering an osteogenicallyeffective amount of activin alone, or in combination with BMPs and/orbone marrow.

BACKGROUND OF THE INVENTION

Activins are dimeric proteins structurally similar to inhibin, TGF-β1,TGF-β2, and other proteins that makeup a family of proteins structurallyrelated to TGF-β1. These proteins exhibit the chromatographic propertiesof TGF-βs. In addition to having homology with respect to the amino acidsequences, activins exhibit conservation of cysteine positionscharacteristic of the TGF-βs. Activins exhibit a molecular weight of 25kD under nonreducing conditions by SDS-PAGE (and a molecular weight of14 kD under reducing conditions). There are two known forms of theactivin subunits, which have been termed βA or βB. Homodimeric forms βAAand βBB and a heterodimeric form βAB have been described in theliterature. Activin subunits have about a 30% homology to TGF-β1 andTGF-β2 chains in terms of their amino acid sequences. Inhibins arepolypeptides which are also structurally related to activins. Inhibinsare heterodimers of the activin βA or βB subunit and a separate αsubunit. Inhibins exhibit activity essentially opposite to activin.

The activin βA homodimer and βAB heterodimer have been purified fromporcine ovarian follicular fluid, and have been shown to stimulate therelease of follicle stimulating hormone (FSH) from rat pituitary cellsin vitro (W. Vale et al., Nature (1986) 321:776-79). Other reportedactivities include stimulation of oxytocin release from neurosecretoryneurons (P. E. Sawchemko, et al., Nature (1988) 334:615-17; W. Vale etal., "Recent progress in Hormone Research" (1988) 44:1-34); stimulationof insulin secretion from pancreatic islets (Y. Totsuka et al., Biochem.& Biophys. Res. Comm. (1988) 156:335-39); and stimulation of erythroidand multi-potential progenitor cell colony formation in bone marrowculture (J. Yu et al., Nature (1987) 330:765-67; H. E. Broxmeyer et al.,Proc. Natl. Acad. Sci. U.S.A. (1988) 85:9052-56). Activin βA isapparently identical to erythroid differentiation factor (EDF) (M.Murata et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85:2434-38).

Despite the fact that activin is similar in amino acid sequence toTGF-β, activin does not compete with TGF-β for binding to TGF-βreceptors types I, II, or III present on fibroblasts and epithelialcells. However, activin has been reported to compete against binding ofTGF-β1 to rat pituitary tumor cells (S. Cheifetz et al., J. Biol. Chem.(1988) 263:17225-28). TGF-β1 and TGF-β2 have been reported to induceformation of endochondral bone in vivo (M. E. Joyce et al., J. CellBiol. (1990) 110:2195-2207, H. Bentz, et al. (1989) J. Biol. Chem.,264:20805-10).

Although the mRNA encoding activin βA has been detected in severaldifferent tissues, including placenta, pituitary, bone marrow, kidney,spinal cord and brain (H. Meunier et al., Proc. Natl. Acad. Sci U.S.A.(1988) 85:247-51), to date the protein has been isolated only fromporcine ovarian follicular fluid. What has not been reported is theisolation and purification of activin from bone and its ability toinduce bone growth and maturation. Thus, there remains a need for thedevelopment of methodologies to extract activin from bone and to developcompositions and treatment modalities to induce bone growth andmaturation. The present invention offers such.

SUMMARY OF THE INVENTION

Bone growth and maturation are enhanced by administering activin in aformulation and in an amount sufficient for inducing bone tissuedeposition. Preferably, local bone growth and maturation is induced byadministering osteogenically effective amounts of a combination ofactivin and TGF-β, BMPs and/or bone marrow or proteins extractedtherefrom. Systemic bone growth and maturation preferably is induced byadministering osteogenically effective amounts of activin alone, or incombination with TGF-β, BMPs and/or bone marrow or proteins extractedtherefrom. The method of the invention results in the production ofbone, with little or no cartilage.

The present invention further includes compositions for locally inducingthe deposition of bone tissue comprising activin with TGF-β, BMPs,and/or bone marrow or proteins extracted therefrom.

Compositions for systemically inducing the deposition of bone tissuecomprise activin alone, or in combination with TGF-β, BMPs and/or bonemarrow or proteins extracted therefrom.

The compositions include pharmaceutical formulations, which can beadministered locally and systemically, to induce bone growth andmaturation, thereby effectively administered to treat bone disorderssuch as osteoporosis, osteohalisteresis, and osteomalacia.

Another object of the invention is to provide a method for locallyinducing bone growth by administering activin alone or in combinationwith TGF-β, BMP, or bone marrow or proteins extracted therefrom, aloneor in combination, in a dental or orthopedic implant.

Another object is to provide a method for extracting activin from boneand using the activin in a local or systemic treatment method to inducebone growth and maturation.

Another object of the invention is to provide a method for isolating andpurifying activin βAA homodimer from bone tissue.

Another feature of the invention is to regulate bone growth andmaturation by first administering an effective amount of TGF-β and BMPsto induce bone formation, and secondly administering an effective amountof activin to induce bone maturation.

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the isolation, purification, formulation and useas more fully set forth below, reference being made to the accompanyingdrawings forming a part hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of dose response curves regarding the release of FSHas described in Example 2.

FIG. 2 is a graph of the results obtained in an alkaline phosphataseassay at 14 days, as described in Example 3.

FIG. 3 is a histological graph of the results described in Example 4.

FIG. 4 is a graph of the results obtained in an alkaline phosphataseassay at 14 days, as described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION A. Overview

It must be noted that as used in this specification and the appendedclaims, the singular forms "a", "an" and "the" include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to "an activin" includes statistical mixtures of dimericproteins of the type generally described herein; reference to "a bonemorphogenetic protein" includes statistical mixtures of such proteins ofthe type described herein; and reference to "the method ofadministration" includes one or more methods of the type describedherein and/or of the type which will become apparent to those ofordinary skill in the art upon reading this disclosure.

In general, the invention involves a method of inducing the depositionof mature bone tissue by administering an osteogenically effectiveamount of activin to a vertebrate subject in need of increased bone. Theactivin may be administered by itself or in combination with anadditional growth factor such as TGF-β, BMP, or with bone marrow and/orproteins extracted therefrom. In accordance with one method of use,activin is administered (in combination with other growth factors and/orbone marrow) locally to the specific area in need of bone growth.Another method of treatment is carried out by administering an activinformulation systemically, thereby systemically inducing bone growth. Inpreferred embodiments, for local administration, activin in combinationwith BMPs and/or bone marrow, or proteins extracted therefrom, isadministered in pharmaceutically acceptable excipients. For systemicadministration, activin alone, or in combination with BMPs and/or bonemarrow, or proteins extracted therefrom, is administered inpharmaceutically acceptable excipients.

Bone growth and maturation may be regulated by first administering aneffective amount of TGF-β and BMPs in pharmaceutically acceptableexcipients, to induce massive bone growth, and secondly administering anosteogenically effective amount of activin, in pharmaceuticallyacceptable excipients, to induce bone maturation. Alternatively, bonegrowth and maturation may be regulated by administering effectiveamounts of TGF-β, BMPs, and activin, in pharmaceutically acceptableexcipients.

The activin alone or in combination with other growth factors may beadministered on a daily basis for 1-14 days, and more preferably 3-10days, and most preferably about 7 days. The amount of activin alone orin combination with other growth factors which is administered will varydepending upon the size and particular needs of the patient. Therefore,the amount administered and the frequency and length of administrationwill be determined by the care giver depending on the needs and thepatient responsiveness with respect to the particular formulation beingadministered. In general, the ratio of BMP to activin will be about1:0.01 to about 1:100 by weight. When activin is used in combinationwith bone marrow, the ratio of activin to bone marrow will be about0.001 μg:1 g to about 100 mg:1 g by weight.

B. Definitions

The term "activin" as used herein refers to activin βAA, activin βAB,activin βBB, and fragments thereof, synthetic peptides, and proteinshaving similar activity in a standard cell culture assay where theprotein stimulates the release of follicular stimulating hormone (FSH)from rat pituitary cells (Vale, et al., Nature (1986) 321:776). Briefly,in this assay, anterior pituitaries from adult male Sprague-Dawley rats(200-220 g) are dissociated by collagenase and plated at a concentrationof 0.33×10⁶ cells per well in 24 well tissue culture dishes. The cellsare allowed to recover for 72 hr in medium containing 2% fetal bovineserum (FBS). Following the recovery period, the cells are washed twicein fresh medium containing 2% FBS. All treatments are added at this timeand the cells are incubated for 72 hr. The media is then collected andthe FSH levels are determined using a radioimmunoassay kit provided bythe National Hormone and Pituitary program of NIADDK.

For purposes of bone induction, activin may be isolated from naturalsources or prepared by recombinant methods (R. H. Schwall et al., Mol.Endo. (1988) 2:1237-42). The primary sequence of activin is highlyconserved from species to species (the activin βA chain is identical inhuman, porcine, bovine, and rat: Vale et al., Recent Progress in HormoneResearch (1988) 44:1-34), as is the case with the TGF-βs. Thus, forexample, it is expected that activin obtained from any vertebrate sourcewill be useful in any vertebrate subject.

The term "BMPs" refers to a bone morphogenetic protein, or proteinsisolated from bone, and fragments thereof and synthetic peptides whichare capable of inducing bone deposition alone or when combined withappropriate cofactors such as TGF-β (or activin). Preparation of BMPs isdescribed in PCT/US87/01537, publication number WO 88/00205, which isincorporated herein by reference to disclose BMPs -1, -2, -3, and -4,and/or their method of administration. J. M. Wozney, in Growth FactorResearch, Vol. 1 (1989), pp 267-280 describes three additional BMPproteins closely related to BMP-2, and which have been designated BMP-5,-6, and -7. WO 89/09787 and 89/09788 describe a protein called "OP-1,"now known to be BMP-7. The cloning of BMP-7 is described in E. Ozkaynaket al., EMBO Journal (1990) 9:2985-2093, and the purification of BMP-7is described in T. K. Sampath et al., J. Biol. Chem. (1990)265:13198-13205.

The BMPs may also include proteins that induce elevated levels of boneformation when combined with appropriate growth factors such as TGF-β oractivin.

The term "TGF-β" refers to beta-type transforming growth factors,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, heterodimers of theTGF-β polypeptide chains (e.g. TGF-β1.2), and fragments thereof,synthetic peptides, and homologous proteins (having substantiallyequivalent biological activity in the TGF-β assay described in Methodsfor Preparation of Media, Supplements, and Substrate for Serum-freeAnimal Cell Culture (1984) pp. 181-194. Alan R. Liss, Inc.). The assaydetermines ability to induce anchorage-dependent growth innon-neoplastic normal rat kidney (NRK) fibroblasts by measuring theformation of cell colonies in soft agar. Preparation of TGF-β1 andTGF-β2 is described in U.S. Pat. No. 4,774,322, incorporated herein byreference. Additional TGF-βs have also been described. U.S. Pat. No.4,886,747 describes the identification of TGF-β3 and its nucleotidesequence, and describes a method for recovery of TGF-β from recombinantcell cultures. S. B. Jakowlew et al., Molec. Endocrinol. (1988)2:1186-1195, describes TGF-β4 and its nucleotide sequence, identified bycDNA characterization. A. B. Roberts et al., Growth Factors, Vol. 2(1990) pp. 135-147, describes the purification of TGF-β5 fromXenopus-conditioned medium.

The term "bone growth" relates to bone mass. TGF-β is thought toincrease bone mass systemically. This is suggested by the increase inthe number and size of osteoblasts, and increased deposition of osteoidlining bone surfaces following systemic administration.

The term "mature bone" relates to bone that is mineralized, in contrastto non-mineralized bone such as osteoid. Administration of activinresults in a stimulation of mature, mineralized bone formation whereasTGF-β preferentially stimulates formation of new osteoid.

The term "osteogenically effective" means that amount which effects theformation and development of mature bone.

The term "subject" as used herein refers to a living vertebrate animalsuch as a mammal or bird in need of treatment, i.e., in need of boneinduction. Such need arises in cases of bone fracture, nonunion, defect,prosthesis implantation, and the like. Such need also arises in cases ofsystemic bone disease, as in osteoporosis.

The term "treatment" as used herein shall mean (1) providing a subjectwith an amount of a substance sufficient to act prophylactically toprevent the development of a weakened and/or unhealthy state; or (2)providing a subject with a sufficient amount of a substance so as toalleviate or eliminate a disease state and/or the symptoms of a diseasestate, and a weakened and/or unhealthy state.

C. General Methods

BMPs and TGF-βs are prepared by methods known in the art (see e.g.,PCT/US87/01537 and U.S. Pat. No. 4,774,322 which are incorporated hereinby reference to disclose such), or are available from commercial sources(R&D Systems, Minneapolis, Minn.). Activin may be isolated fromfollicular fluid, prepared by recombinant methods, or isolated from boneusing the methods disclosed below.

Activin is similar in hydrophobicity to TGF-β1 and TGF-β2 (but has alower pI than these polypeptide factors) and is isolated from bone in asimilar fashion. J. M. Vaughan et al., Meth. Enzymol. (1989)168:588-617, describes a method for preparing anti-activin βA antibody.It is well known in the art that antibodies are useful in isolation andpurification procedures. Alternatively, a detailed isolation procedurein accordance with the present invention is described in Example 1below. The characterization of activin is described in Example 2 below.

Pharmaceutical formulations of the invention which include activin foradministration will generally include an osteogenically effective amountof activin, and optionally, BMP, to promote bone growth, in addition toa pharmaceutically acceptable excipient. Suitable excipients includemost carriers approved for parenteral administration, including water,saline, Ringer's solution, Hank's solution, and solutions of glucose,lactose, dextrose, ethanol, glycerol, albumin, and the like. Thesecompositions may optionally include stabilizers, antioxidants,antimicrobials, preservatives, buffering agents, surfactants, and otheraccessory additives. A presently preferred vehicle comprises about 1mg/ml serum albumin (species-specific) in phosphate-buffered saline(PBS). A thorough discussion of suitable vehicles for parenteraladministration may be found in E. W. Martin, "Remington's PharmaceuticalSciences" (Mack Pub. Co., current edition sections relating to theexcipient vehicles and formulating being incorporated herein byreference to disclose such). Such formulations are generally known tothose skilled in the art and are administered systemically to providesystemic treatment.

Compositions of the invention may also be implanted directly at the siteto be treated, for example, by injection or surgical implantation of thecomposition in a sustained-release carrier. Suitable carriers includehydrogels, controlled- or sustained-release devices (e.g., an Alzet®minipump), polylactic acid, and collagen matrices. Presently preferredcarriers are formulations of atelopeptide collagen containingparticulate calcium phosphate mineral components, such as combinationsof fibrillar atelopeptide collagen (for example Zyderm® CollagenImplant, available from Collagen Corporation, Palo Alto, Calif.) withhydroxyapatite-tricalcium phosphate (HA-TCP, available from Zimmer,Inc., Warsaw, Ind.). It is presently preferred to administer implantcompositions containing activin and BMP in a collagen/mineral mixtureimplant.

The proteins of the invention may be conjugated to other molecules toincrease their water-solubility, increase their half-lives, or enhancetheir ability to bind to bone. For instance, they may be conjugated topolyethylene glycol to increase their water solubility or tobone-binding molecules such as bisphosphonates (e.g.1-hydroxyethylidene-1,1-bisphosphonic acid, dichloromethylenebisphosphonic acid, and 3-amino-1-hydroxypropylidene-1-bisphosphonicacid) and fluorochromes (e.g. tetracyclines, calcein blue, xylenolorange, calcein green, and alizarin complexone red) to target theproteins to bony sites. Various agents for conjugating molecules toproteins are well known in the art and include aldehydes, carbodiimides,and other bifunctional moieties.

The precise dosage necessary will vary with the age, size, sex andcondition of the subject, the nature and severity of the disorder to betreated, and the like; thus, a precise effective amount cannot bespecified in advance and will be determined by the care giver. However,appropriate amounts may be determined by routine experimentation withanimal models, as described below. In general terms, an effective doseof activin for systemic treatment will range from about 0.001 μg/kg toabout 10 mg/kg of body weight. An effective dose for BMP is about 0.001μg/kg to about 10 mg/kg of body weight.

An effective dose for activin for local treatment will range from about0.001 μg/kg to about 10 mg/kg of body weight. An effective dose of BMPfor local treatment is substantially the same as the dose of activin.

In addition, it may be desirable to combine the proteins with othertherapeutics, such as, for instance in the case of osteoporosis,fluoride, calcitonin, vitamin D metabolites, estrogen, and parathyroidhormone. Because proteins are non-species-specific in their activitythey maybe used to treat subjects in general, including sport, pet, andfarm animals, and humans.

Suitable animal models for bone growth and maturation include the mousemodel as illustrated in Examples 3, 4 and 5 below. Briefly, systemictreatment is assayed by injecting test amounts of activin into a mouseor other experimental animal following a standard protocol, followed byexamination of bone strength and mass at approximately 14 dayspost-administration. Such examination may be performed in situ by usingimaging techniques (e.g., nuclear magnetic resonance imaging, X-raytomography, ultrasound, and sound conduction) or stress testing, or exvivo by standard histological methods. Suitable dosages and protocolswill result in improved deposition of mature bone, for example,increased cortical thickness in long bones.

Compositions of the invention are useful for treating bone fractures,defects, and disorders which result in weakened bones such asosteoporosis, osteohalisteresis, osteomalacia, and age-related loss ofbone mass. Compositions of the invention that are delivered insustained-release vehicles are also particularly useful for improvingimplant fixation, for example for improving ingrowth of new bone into ametal prosthesis in joint reconstruction and dental or orthopedicimplants.

Dental and orthopedic implants can be coated with osteoinductiveproteins, such as BMP, activin, TGF-β, and bone marrow or proteinsextracted therefrom, either in combination or alone, to enhanceattachment of the implant device to the bone. Implant devices generallyhave a porous end, which is inserted into the implant site. The porousend allows the new bone to grow into the porous space and hold theimplant in place. The greater the growth of new bone into the porousspace, more solidly the implant is held in place. The presence ofosteoinductive proteins, such as BMP, activin, and TGF-β, and bonemarrow or proteins extracted therefrom, either alone or in combination,in the porous space is expected to enhance the growth of new bone intothe porous space.

In general, implant devices may be coated with osteoinductive proteinsas follows. Activin, BMP, and TGF-β, and bone marrow or proteinsextracted therefrom, either alone or in combination, are dissolved at aconcentration in the range of 0.01 μg/ml to 200 mg/ml inphosphate-buffered saline (PBS) containing 2 mg/ml serum albumin. Theporous end of an implant is dipped in the solution and is airdried orimplanted immediately into the bony site. The viscosity of the coatingsolution is increased, if desired, by adding hyaluronate at a finalconcentration of 0.1 mg/ml to 100 mg/ml or by adding otherpharmaceutically acceptable excipients. Alternatively, the solutioncontaining the osteoinductive factor is mixed with collagen gel (e.g.Zyderm® Collagen Implant, Collagen Corp., Palo Alto, Calif.) to a finalcollagen concentration of 5 mg/ml to 100 mg/ml to form a paste, which isthen used to coat the porous end of the implant device. The coatedimplant device is placed into the bony site immediately or is airdriedand rehydrated with PBS prior to implanting.

Either native or synthetic (recombinant) activin can be used to produceantibodies, both polyclonal and monoclonal. The term "antibody" isintended to include whole immunoglobulin of any isotype or species aswell as antigen binding fragments and chimeric constructs. If polyclonalantibodies are desired, purified activin is used to immunize a selectedmammal (e.g. mouse, rabbit, goat, horse, etc.) and serum from theimmunized animal later collected and treated according to knownprocedures. Compositions containing polyclonal antibodies to a varietyof antigens in addition to activin can be made substantially free ofantibodies which are not anti-activin antibodies by passing thecomposition through a column to which activin has been bound. Afterwashing, polyclonal antibodies are eluted from the column. Monoclonalanti-activin antibodies can also be readily produced by one skilled inthe art. The general methodology for making monoclonal antibodies byhybridomas is well known. Immortal, antibody-producing cell lines canalso be created by techniques other than fusion, such as directtransformation of B lymphocytes with oncogenic DNA, or transfection withEpstein-Barr virus, M. Schrier Hybridoma techniques (1980); Hammerlinget al., Monoclonal Antibodies and T cell Hybridomas (1981); Kennett etal., Monoclonal Antibodies (1980).

By employing activin (native or synthetic) as an antigen in theimmunization of the source of the B cells immortalized for theproduction of monoclonal antibodies, a panel of monoclonal antibodiesrecognizing epitopes at different sites on the activin molecule can beobtained. Antibodies which recognize an epitope in the binding region ofthe protein can be readily identified in competition assays betweenantibodies and proteins. Antibodies which recognize a site on theactivin protein are useful, for example, in the purification of theprotein from cell lysates or fermentation media, in the characterizationof the protein, and in identifying immunologically related proteins.Such immunologically related proteins (i.e. that exhibit common epitopeswith activin) are another aspect of the invention. In general, as it isknown in the art, the anti-activin antibody is fixed (immobilized) to asolid support, such as a column or latex beads, contacted with asolution containing activin, and separated from the solution. Theactivin, bound to the immobilized antibodies, is then eluted.

Methods for inducing local bone growth and maturation compriseadministering an osteogenically effective amount of activin, incombination with BMPs, bone marrow, or proteins extracted therefrom, ina pharmaceutically acceptable carrier, as describe in Example 3, below.

Methods for inducing systemic bone growth and maturation comprisesadministering an osteogenically effective amount of activin alone, andoptionally including BMPs, bone arrow, or proteins extracted therefrom,in a pharmaceutically acceptable carrier, as described in Example 4,below.

Methods for regulating bone growth and maturation comprises initiallyadministering an effective amount of TGF-β and BMPs, and sequentiallyadministering an effective amount of activin. Preferably, to induceendochondral bone growth, combinations of BMP and TGF-β may be locallyadministered to induce cartilage modeling. Activin may be subsequentlyadministered subcutaneously and/or systemically to induce mature boneformation and differentiation of the cartilage model induced by TGF-βand BMP. The activin would enhance mineralization of the endochondralbone and maturation. It is expected that the quantity of mature boneformed by this method would be greater than the quantity of mature boneformed by either (i) TGF-β, BMPs, or activin alone, or (ii) combinationsof TGF-β and BMPs, or (iii) combinations of activin and BMPs.

Alternative methods for regulating bone growth and maturation comprise(i) locally administering an osteogenically effective amount of activinwith TGF-β, BMP and/or bone marrow or proteins extracted therefrom, toinduce bone growth and maturation, or (ii) systemically administeringeffective amounts of TGF-β, BMP and/or bone marrow or proteins extractedtherefrom to induce bone growth, followed by systemically administeringan osteogenically effective amount of activin to induce bone maturation.

Activin is extracted from demineralized bone powder with 6M guanidineHCl, 10 mM EDTA, pH 6.8. The extract is fractionated by gel filtrationchromatography. The pool of fractions containing TGF-β is desalted andfractionated by cation exchange chromatography. The pool of fractionscontaining activin is then fractionated by C18 RP-HPLC, using a linearacetonitrile gradient in 0.1% trifluoroacetic acid (TFA). Activin elutesbetween TGF-β1 and TGF-β2. The activin fraction is applied onto a cationexchange Mono-S FPLC column (Pharmacia) at pH 4.6. The column isequilibrated sequentially into pH 6.7 and pH 9.0 buffers. Activin, whichelutes during the pH 6.7-9.0 gradient, is desalted by C18 RP-HPLCperformed in 0.1% TFA with a linear acetonitrile gradient as the finalpurification step.

Since TGF-β1 and TGF-β2 have similar chromatographic properties asactivin, activin preparations may be contaminated with low levels ofTGF-β. The majority of the contaminating TGF-β activity may be removedduring the pH 4.6-6.7 gradient performed on the Mono-S FPLC, resultingin substantially pure preparations of activin. In addition, the majorityof TGF-β2 elutes slightly later than activin during the pH 6.7-9.0gradient, while TGF-β1 is retained on the column during the Mono-S FLPCstep. The use of a two step salt gradient, as compared to standardtechniques using a single step salt gradient, is a uniquechromatographic procedure which results in significantly purepreparations of activin.

D. Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toextract, isolate, formulate and use the compositions and methods of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to insure accuracywith respect to numbers used (e.g., amounts, times, temperature, etc.),but some experimental error and deviations should be accounted for.Unless indicated otherwise, parts are parts by weight, temperature is indegrees centigrade, pressure is at or near atmospheric, and otherparameters are conventional and in accordance with those normallyaccepted by those skilled in the art.

EXAMPLE 1 Extraction and Isolation of Activin

Activin βAA was isolated and purified from 75 kg of bovine bone powderusing the procedure described below. The extract of demineralized bonepowder was prepared from fresh metatarsal and metacarpal bones asdescribed by Seyedin et al., U.S. Pat. No. 4,774,322.

Crude bone extract in 6M guanidine HCl, 10 mM EDTA, pH 6.8, wasfractionated by column chromatography on Sephacryl® S-200, and thefractions containing high levels of TGF-β were pooled. The TGF-β poolwas concentrated by ultrafiltration and was desalted by passage throughan Amicon GH-25 column equilibrated in 6M urea, 50 mM sodium acetate, 10mM NaCl, pH 4.6.

The NaCl concentration of the TGF-β pool was increased to 70 mM, and theproduct applied onto a Whatman® CM-52 column (2.5×38 cm) equilibrated ina buffer of 6M urea, 50 mM sodium acetate, 70 mM NaCl, 1% isopropanol,pH 4.6. The urea/acetate buffer was pumped through the column until A₂₈₀returned to the baseline. The bound protein was eluted using a linear70-600 mM NaCl gradient (1000 ml total volume) in the urea/acetatebuffer at a flow rate of 30 ml/hour. The CM-52 fractions were analyzedby SDS-PAGE (U. Laemmli, Nature (1970) 227:680-85) on a 15%polyacrylamide gel, and three separate CM-bound pools were made. Pools 2(fractions 61-70) and 3 (fractions 71-83) contained the majority ofTGF-β2 and TGF-β1, respectively. Pool 1, which consisted of fractions55-60, contained the majority of activin.

Each of the pools was individually chromatographed on a C18 RP-HPLCcolumn (1×25 cm, Vydac, 218TP510) equilibrated in 0.1% trifluoroaceticacid (TFA). The bound protein was eluted from the column using a lineargradient of acetonitrile from 32-52% buffer B (90% acetonitrile, 0.1%TFA) over 20 minutes at a flow rate of 3 ml/minute. Fractions werecollected manually in order to isolate individual peaks, and thefractions were analyzed by SDS-PAGE under nonreducing conditions. A 25kD protein (activin) migrating slightly faster than TGF-β on anSDS-polyacrylamide gel was observed to elute from the reverse-phase HPLCcolumn between the elution positions of TGF-β1 and TGF-β2 in theCM-bound pools 1 and 2. The CM-bound pool 3, which contained themajority of the TGF-β1, was later shown to contain very little activin,and no further attempts were made to isolate it from pool 3.

The 25 kD protein (activin) fractions from the CM-bound pools 1 and 2were combined. The pool was diluted 3-fold with 6M urea, 50 mM sodiumacetate, 10 mM NaCl, 1% isopropanol, pH 4.6, and was loaded onto aMono-S FPLC column (Pharmacia HR5/5), which was equilibrated in the samebuffer. Some of the bound proteins eluted when the pH was raised from4.5 to 6.7 by equilibrating the column into 6M urea, 50 mM sodiumacetate, 10 mM NaCl, 1% isopropanol, pH 6.7 at a flow rate of 0.5 ml/minover a 10 minute period. The column was then equilibrated into 6M urea,20 mM HEPES, 10 mM NaCl, 1% isopropanol, pH 6.7, and the 25 kD protein(activin) was eluted by equilibrating the column into 6 mM urea, 20 mMHEPES, 10 mM NaCl, 1% isopropanol, pH 9.0, at a flow rate of 0.5 ml/minover a 10 min period.

A linear NaCl gradient from 10-400 mM NaCl in urea/HEPES buffer, pH 9.0,at a flow rate of 0.5 ml/min over a 10 min period, was then used toelute remaining bound proteins. The SDS-polyacrylamide gels were stainedwith silver (J. H. Morrisey, Anal. Biochem. (1981) 117:301-310). Themajority of TGF-β activity, which would otherwise contaminate activin,was removed by performing the pH 4.7-6.7 gradient. Activin eluted duringthe pH 6.7-9.0 gradient. The elution position of activin is slightlybefore that of TGF-β2 during the pH 6.7-9.0 gradient. TGF-β1 elutesduring the 10-400 mM NaCl gradient at pH 9.0.

Some 25 kD TGF-β-like protein (including TGF-β1) eluted during the NaClgradient and was highly contaminated with low molecular weight proteins(12 kD to 18 kD).

The 25 kD protein (activin) was chromatographed on a C18 RP-HPLC columnas the final purification step. The pool of 25 kD protein fractions fromthe pH 6.7-9.0 gradient of the Mono-S chromatography was acidified byadding glacial acetic acid to a final concentration of 1.0M, and wasapplied to a C18 RP-HPLC analytical column (Vydac, 4.6×250 mm, 218TP54)equilibrated in 0.1% TFA. The column was run at a flow rate of 1 ml/min,and the bound protein was eluted using a linear acetonitrile gradientfrom 32-52% buffer B at a rate of 1% buffer B/min. Fractions wereanalyzed by SDS-PAGE under nonreduced conditions. The activin fractiondemonstrated one single major band on the silver-stainedSDS-polyacrylamide gel. No other bands were seen. The total yield of the25 kD protein (activin) from the CM-bound pools 1 and 2 wasapproximately 1.2 mg, corresponding to 16 μg/kg bone powder.

EXAMPLE 2 Identification of the 25 kD Protein as Activin

A) The N-terminus of the 25 kD protein obtained in Example 1 above wassequenced on an Applied Biosystems sequencer. The first 36 amino acidswereGly-Leu-Glu-(?)-Asp-Gly-Lys-Val-Asn-Ile-(?)-(?)-Lys-Lys-Gln-Phe-Phe-Val-Ser-Phe-Lys-Asp-Ile-Glu-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-Ser-Gly-Tyr-His(where "?" denotes an unidentified amino acid). This sequencecorresponds to the first 36 amino acid residues at the N -terminus ofthe activin βA subunit, in which each of the "?"s is Cys (Cys is notdetected in the sequencing process). No other amino acid sequences wereobserved. The entire activin βA subunit sequence, in single letter code,is ##STR1## (W. Vale et al., Recent Progress in Hormone Research (1988)44:1-34.

(B) A 5 μg sample of the activin obtained in Example 1 above was reducedand denatured by incubating for 2 hours at 50° C. under N₂ atmosphere in250 μL of alkylation buffer (0.4M Tris/HCl, pH 8.5, 6M urea, 0.1% EDTA,20 mM DTT). The reduced protein was alkylated by incubating in thepresence of 100 mM iodoacetamide at room temperature in the dark for 4hrs. The sample was subjected to SDS-PAGE.

The silver-stained gel of reduced and alkylated 25 kD protein exhibiteda single band migrating at a molecular weight of 14 kD. This observationis consistent with the 25 kD protein being activin, where activin isknown to be a dimer of two 14 kD subunits.

(C) The activin extracted and isolated in Example 1 above was assayedfor biological activity by testing the ability to stimulate release offollicle-stimulating hormone (FSH) by rat pituitary cells in culturefollowing the method of W. Vale et al., Endocrinology (1972) 91:562-572.Briefly, anterior pituitaries from adult male Sprague-Dawley rats(200-220 g) were dissociated by collagenase and plated at aconcentration of 0.33-106 cells per well in 24-well tissue culturedishes. The cells were allowed to recover for 72 hours in mediumcontaining 2% fetal bovine serum (FBS). Following the recovery period,the cells were washed twice in medium containing 2% FBS. All treatmentswere added at this time and the cells were incubated for 72 hr. Themedia was then collected and FSH levels determined using aradioimmunoassay kit provided by the National Hormone and PituitaryProgram of NIADDK. The protein isolated in Example 1 exhibitedFSH-releasing activity equal to activin derived from porcine follicularfluid. TGF-β1 and TGF-β2 did not exhibit FSH-releasing activity, asexpected. The results are shown in FIG. 1. The results described in (A),(B) and (C) demonstrate that the 25 kD protein purified from bone inExample 1 is activin βA homodimer.

The activin purified from bovine bone, as described in Example I, wasassayed for the protein's ability to stimulate formation of erythroidcolonies following the method of Broxmeyer et al., Proc. Natl. Acad.Sci. U.S.A. (1988) 85:9052-6. Single cell suspensions of normal C57B1/6male murine femur bone marrow and spleen were prepared for in vitroassay. Activin stimulated increases in the colony-forming uniterythroids (CFU-E) and burst-forming unit erythroid (BFU-E) in adose-dependent fashion in the similar range of activin concentration asthose reported for recombinant human activin (Broxmeyer et al., Proc.Natl. Acad. Sci. U.S.A. (1988) 85:9052-6). Activin also significantlystimulated both the CFU-E and BFU-E of spleen cells. The results aresummarized in Table I below.

                  TABLE ONE                                                       ______________________________________                                        Effects of activin on erythroid colony formation                              Bone marrow (BM)                                                              ______________________________________                                                   CFU-E/     %               %                                                  10 5       CON-    BFU-E/  CON-                                    GROUP      BM         TROL    10 5 BM TROL                                    ______________________________________                                        Control    215 ± 38                                                                              100     10 ± 2                                                                             100                                     Activin 100 ng/ml                                                                        309 ± 32**                                                                            143     19 ± 1**                                                                           188                                     50 ng/ml   296 ± 15                                                                              138     16 ± 2*                                                                            163                                     25 ng/ml   269 ± 25                                                                              125     16 ± 4*                                                                            155                                     12.5 ng/ml 245 ± 51                                                                              114     15 ± 4                                                                             150                                     6.25 ng/ml 239 ± 22                                                                              111     13 ± 3                                                                             125                                     3.12 ng/ml 229 ± 20                                                                              106     10 ± 2                                                                             100                                     1.56 ng/ml 215 ± 34                                                                              100     10 ± 2                                                                             100                                     ______________________________________                                                              %               %                                       Spleen     BFU-E/10.sup.5                                                                           control CFU-E/10.sup.5                                                                        control                                 ______________________________________                                        control    2.4 + 0.4  100     255 + 16                                                                              100                                     50 ng/ml     7 + 1    271     429 + 20                                                                              168                                     activin                                                                       ______________________________________                                    

EXAMPLE 3 Local Induction of Bone Growth and Maturation

The following experiment demonstrated induction of bone growth in vivousing a combination of BMP and activin.

A ceramic/collagen carrier was prepared using hydroxyapatite, tricalciumphosphate, and soluble bovine dermal collagen (VITROGEN®collagen-in-solution, Collagen Corporation) as described by H. Bentz etal., J. Biol. Chem. (1989) 264:20805-810. BMP was prepared as describedin E. A. Wang et al., Proc. Natl. Acad. Sci. USA (1988) 85:9484-88.Activin was obtained as described in Example 1 above.

Compositions were prepared as follows:

A: Carrier alone

1: BMP (1 μg)

2: BMP (1 μg), TGF-β2 (7.5 μg)

3: BMP (1 μg), TGF-β2 (2.5 μg)

4: BMP (1 μg), activin (7.5 μg)

5: BMP (1 μg), activin (2.5 μg)

6: BMP (1 μg)+TGF-β2 (7.5 μg) +activin (7.5 μg).

7: activin (2.5 μg)

The compositions were then implanted at the ectopic subcutaneous sitesin the ventral thorax region of male Sprague-Dawley rats at 6rats/composition (except for carrier alone, BMP alone, and activinalone, at 4 rats/composition. After 14 days, the implants were excised,evaluated biochemically for alkaline phosphatase activity (Bentz et al.,J. Biol. Chem. (1989) 264:20805-20810; Reddi et al., Proc. Natl. Acad.Sci. U.S.A. (1972) 69:1601-5) and examined histologically for bonegrowth. The biochemical results are shown graphically in FIG. 2.

Biochemistry

Implants containing BMP or activin alone exhibited very low alkalinephosphatase activity. (See columns 1 and 7 in FIG. 2.) The compositionscontaining BMP and either TGF-β2 or activin or both (at either dosage)exhibited high levels of alkaline phosphatase activity.

Histology:

Compositions containing carrier alone, or carrier with activin,exhibited no bone growth, and displayed mild to moderate foreign bodyreactions. Compositions containing BMP alone exhibited low to moderatelevels of bone and no cartilage.

Compositions containing BMP and TGF-β2 exhibited a large increase inboth bone and cartilage. Composition 3 (2.5 μg TGF-β2) exhibited morebone, and more differentiation of the bone tissue, than composition 2(7.5 μg TGF-β2). Composition 2 induced more cartilage than bone.

Compositions containing BMP and activin (4 and 5) exhibited little or nocartilage, and significantly elevated levels of deposited mature bone.Composition 5 (2.5 μg activin) induced deposition of more bone thancomposition 6 (7.5 μg activin). However, both compositions induced verylittle cartilage deposition, and produced more mature bone than thatobtained using BMP with TGF-β2.

Composition 6 (BMP, TGF-β2, and activin) induced a poorlydifferentiated, disorganized mixture of bone, fibrocartilage, andfibrosis.

The experimental results demonstrated that activin functions as anosteogenic cofactor for BMP in vivo, and induces growth of high-qualitymature bone, without inducing cartilage formation.

EXAMPLE 4 Systemic Administration of Activin

The following experiment demonstrated the activity of activin uponsystemic administration in vivo.

Neonatal Mice

Neonatal mice (Swiss-Webster, 8-10 litters per group) were given dailyadministrations of:

1: 1 μg TGF-β1

2: 1 μg activin

3: 3 μg activin

4: 1 μg TGF-β1+1 μg activin

5: vehicle alone in a vehicle of 37.5 μg mouse serum albumin (MSA) in 25μl PBS. The control group received vehicle alone. The compositions wereadministered daily for 4 days, by subcutaneous injection in the nape ofthe neck. The femurs were harvested on the fifth day, and examinedhistologically.

The mice receiving 1 μg/day activin exhibited superior bone growth andmaturation, displaying reduced porosity in the femoral shaft comparedwith controls. In contrast, mice receiving 1 μg/day TGF-β1 exhibitedincreased porosity compared with controls. Both groups exhibitedincreased levels of osteoclasts, and hypertrophy of osteoblasts relativeto controls, suggesting increased bone remodeling. However, the groupreceiving 3 μg/day activin exhibited decreased levels of osteoclasts,and flat, inactive-appearing osteoblasts. Administration of activin andTGF-β1 together inhibited the changes in osteoclasts and osteoblasts,and did not increase femoral shaft porosity. The results are graphicallyshown in FIG. 3.

The results indicate that systemic administration of activin for alimited treatment course improves bone growth and maturation by inducingbone formation and promoting bone remodeling.

EXAMPLE 5 Activin Enhances Osteogenesis in Bone Marrow

Implantation of bone marrow in collagen/hydroxyapatite-tricalciumphosphate ceramic carrier in rat subcutis results in local boneformation in the ceramic implant at ectopic sites. This process, knownas osteogenesis, is probably due to differentiation of a specific cellpopulation in the bone marrow. Activin, when added to the bone marrow,promotes greater mature bone deposition.

Young adult male Lewis rats (8 weeks old, 3 rats/group) were implantedsubcutaneously in the ventral thorax region with ceramic carriercontaining 25 or 50 μl of rat bone marrow plus 5 μg of activin orTGF-β2. After 14 days, the implants were excised, evaluated for alkalinephosphatase activity and examined histologically for bone formation.

Ceramic carrier alone had almost no alkaline phosphatase activity andhistologically, exhibited low levels of inflammatory cells and no bone.Ceramic carrier with 20 μl of bone marrow exhibited low levels of boneformation. However, the group with 20 μl of bone marrow plus activinshowed elevated levels of bone formation over the levels observed with20 μl of bone in the implants of bone marrow alone and bone marrow plusactivin when compared with the carrier alone (FIG. 4). In a secondstudy, 10 μl of bone marrow plus TGF-β2 showed less bone and a higherlevel of fibrosis than the group with bone marrow plus activin. Althoughthe alkaline phosphatase activity was elevated, it was less than thelevel observed with bone marrow plus activin. These experimental resultsdemonstrate that activin uniquely enhances bone formation with theabsence of fibrosis when administered with bone marrow.

The present invention is shown and described herein and was consideredto be the most practical, and preferred embodiment. It is recognized,however, that departures may be made therefrom which are within thescope of the invention and that obvious modifications will occur to oneskilled in the art upon reading this disclosure.

We claim:
 1. A method for inducing deposition and maturation of bone ina subject in need thereof, which comprises administering anosteogenically effective amount of activin in a pharmaceuticallyacceptable excipient to said subject.
 2. The method of claim 1, whereinthe activin in a pharmaceutically acceptable excipient is administeredsystemically to the subject.
 3. The method of claim 1, wherein theactivin is administered once per day for about 1-14 days.
 4. The methodof claim 1, wherein said subject has osteoporosis.
 5. The method ofclaim 1, wherein the pharmaceutically acceptable excipient comprises amixture of collagen and calcium phosphate.
 6. The method of claim 1,which further comprises administering an effective amount of TGF-β tothe subject.
 7. The method of claim 6, wherein said activin and theTGF-β are administered simultaneously.
 8. The method of claim 6, whereinsaid activin and said TGF-β are administered sequentially.
 9. The methodof claim 1, which further comprises administering an effective amount ofa BMP to the subject.
 10. The method of claim 9, wherein said activinand said BMPs are administered in a ratio of about 1:0.01 to about 1:100by weight.
 11. The method of claim 9, wherein said activin and said BMPsare administered in a ratio of about 1:0.1 to about 1:1 by weight. 12.The method of claim 9, wherein said activin and said BMP administeredsystemically to the subject.
 13. The method of claim 9, wherein saidactivin and said BMP are administered once per day for about 1-14 days.14. The method of claim 9, wherein said subject has osteoporosis. 15.The method of claim 9, wherein said activin and said BMP areadministered simultaneously.
 16. The method of claim 9, wherein saidactivin and said BMP are administered sequentially.
 17. The method ofclaim 9, which further comprises administering an effective amount ofTGF-β to the subject.
 18. The method of claim 1, which further comprisesadministering an effective amount of bone marrow to the subject.
 19. Themethod of claim 18 wherein said activin and said bone marrow areadministered once per day for about 1-14 days.
 20. The method of claim18 wherein said subject has osteoporosis.
 21. The method of claim 18,which further comprises administering an effective amount of TGF-β tothe subject.
 22. The method of claim 18, wherein said activin and saidbone marrow are present in a ratio of about 0.001 μg:1 g to about 100mg:1 g by weight.
 23. The method of claim 18, wherein said bone marrowand said activin are administered in a composition comprising collagenand calcium phosphate.
 24. The method of claim 18, wherein said activinand said bone marrow are administered simultaneously.
 25. The method ofclaim 18, wherein said activin and said bone marrow are administeredsequentially.
 26. The method of claim 3 wherein the activin isadministered once per day for about 3-10 days.
 27. The method of claim26 wherein the activin is administered once per day for about 7 days.28. The method of claim 13 wherein said activin and said BMP areadministered once per day for about 3-10 days.
 29. The method of claim28 wherein said activin and said BMP are administered once per day forabout 7 days.
 30. The method of claim 19, wherein said activin and saidbone marrow are administered once per day for about 3-10 days.
 31. Themethod of claim 30, wherein said activin and said bone marrow areadministered once per day for about 7 days.